Project 1 - The goal of Project 1 is to define the mechanism of action of three new chemokine analogues with potent activity in blocking HIV-1 entry via CCR5. These fully recombinant molecules display three distinct activity profiles: (group I) CCR5 blockade without signaling activity or receptor internalization;(group II), CCR5 rapid internalization with signaling activity;and (group III), moderate CCR5 internalization and blockade without signaling activity. These new molecules have significant potential advantages in terms of safety and cost of production ..over currenynh[b^ fwither, irrtpr.pvejTients.,p,n these bahdidate'microbicides requires more detailed knowledge of their mechanisms of action. We will define the route to intracellular sequestration for group II molecules, and compare with that of the group III molecules. We will examine a number of hypotheses to explain prolonged antiviral activity in the absence of intracellular receptor sequestration (group I) or with moderate internalization (group III), including changes in CCR5 dimer formation, altered receptor localization in the membrane, allosteric effects, and receptor internalization independent of G-protein-linked signaling. We will also examine the impact of CCR5, CCL5, and CCL3L1 genetic polymorphisms on CCR5 protein synthesis and turnover rate. These studies are designed to investigate variability in the susceptibility of primary target cells from normal human donors to each of the new inhibitors. We will also use a panel of mutant CCR5 molecules to examine structural correlates of activity. We will use this information on mechanism and target cell variability to develop new molecules with even better activity profiles, and we will perform coordinated experiments with other projects in this Program to relate our findings in cell-based models to the effects of current and new CCR5 inhibitors in tissue explant and whole animal models. This approach will generate better and safer CCR5 inhibitors that can be produced on a scale suitable for stopping the spread of HIV/AIDS in the most-impacted areas.